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Galanin stimulates phosphatidylinositol turnover in cardiac tissue of the mudpuppy
Journal of the Autonomic Nervous System, 40 (1992) 87-90
87
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01302
Galanin stimulates phosphatidylinositol turnover in cardiac tissue of the mudpuppy Jean C. Hardwick and R o d n e y L. Parsons Department of Anatomy and Neurobiology, College of Medicine, Unit'ersityof Vermont, Burlington, Vermont, USA (Received 17 March 1992) (Revision received 12 May 1992) (Accepted 19 May 1992)
Key words: Galanin; Inositol phosphate; Cardiac muscle; Cyclic AMP; Cyclic GMP Abstract A galanin-like peptide has been found in the parasympathetic fibers innervating the mudpuppy myocardium and direct application of galanin produces hyperpolarization of atrial myocytes and a decrease in twitch tension. In the present study, atrial and ventricular strips were incubated with galanin and then evaluated for changes in either phosphatidylinositol turnover or cyclic nucleotide levels. Galanin caused a significant and concentration-dependent increase in phosphatidylinositol turnover in both atrial and ventricular tissue. However, galanin had no effect on cyclic AMP or cyclic GMP levels in either basal or adrenergically-stimulated preparations. These results suggest that the galanin-induced cardio-inhibition in the mudpuppy may be mediated, at least in part, by a change in phosphatidylinositol turnover.
Introduction
In 1985, Axelsson and Nilsson demonstrated that vagal stimulation of the mudpuppy (Necturus maculosus), in the presence of cholinergic antagonists, produced cardio-inhibition [2]. These authors suggested that another unidentified neurotransmitter, perhaps a neuropeptide, was colocalized with acetylcholine and released from parasympathetic nerve terminals. Subsequently, we demonstrated, both biochemically and histochemically, that a galanin-like peptide is present in postganglionic parasympa-
Correspondence to: R.L. Parsons, Department of Anatomy and Neurobiology, Given Building, University of Vermont, Burlington, VT 05405, USA.
thetic fibers innervating the mudpuppy heart [6,10]. We also observed in isolated atrial and ventricular strips, a galanin-induced decrease in electrically stimulated twitch tension and a decrease in force and rate of spontaneous contractions in isolated septum/sinus venous preparations [10]. Local application of galanin directly onto muscle strands in septal or atrial whole mount preparations or to atrial myocytes hyperpolarized these cells [7,10]. From these results, it appears that a peptide similar to mammalian galanin is the previously unidentified neuropeptide which can produce non-cholinergic cardio-inhibition in the mudpuppy. The alteration in activity of target tissues by neuropeptides is commonly mediated through the activation of intracellular second messengers. Consequently, the present study was undertaken
to determine whether galanin activates second messengers in mudpuppy myocardium which might mediate the galanin-induced cardio-inhibition.
Materials and Methods Mudpuppies were killed by rapid decapitation, the heart removed and the atrial and ventricular regions dissected free. The atria were bisected and the ventricles cut into strips of similar size in a solution containing (in raM): 120 NaCI, 2.5 KCI, 3.6 CaCI 2, 1.0 Hepes, pH 7.3 at 20-22°C. Sections were then randomly distributed such that one or two strips of either atrial or ventricular tissue were loosely pinned in Sylgard-coated wells. To measure phosphatidylinositol turnover, isolated strips were incubated overnight at room temperature in normal Ringer solution containing 0.2 # M 3H-myo-inositol (New England Nuclear, 14-20 C i / m m o l ) to label the phospholipid
pool. Following this incubation, the [issue was washed with several ml of fresh solution and then incubated for 10 min in 1.5 ml of control solution containing 10 mM LiCI to inhibit inositol phosphatase. Galanin (porcine, Peninsula Laborat¢)ries or Sigma Chemical Co.) at concentrations ranging from I0 '~ to 10 +' M was added and the incubation continued for 20 rain at room temperature. The tissue was then immersed in liquid nitrogen, weighed and homogenized in acidified methanol (1: 100) in a ground glass homogenizer. The final volume of 2 ml was centrifuged at 2 0 0 0 x g for 11) min at 4°C and 1.4 ml of the resulting supernate was diluted with 5 ml of water for analysis of inositol phosphates. Total inositol phosphates were isolated by column chromatography, as described by Berridge et al. [3]. Samples were poured over l)owex X 1-8 formate resin and the inositol phosphates collected by elution with 0.1 M formic a c i d / l M ammonium formate solution. The eluate was then counted in a liquid scintillation counter and the
B
A CONIROL
50
tO
-7
100
M GALANIN
I 0 6M GALANIN
40
z o uS
30
c~
80
2D
o
6o
\\, I
I I
20
c3
10
I
"5
40
2~
~
20
x
ATRIA
VENTRICLE
9
!
i
i
-8
-7
-6
log {GALANIN]
Fig. 1. Galanin stimulation of phosphatidylinositol turnover in atrial and ventricular tissue. Atrial and ventricular strips were prelabeled with ~H-myo-inositol and then incubated for 20 rain with either control solution or galanin at the concentration indicated. (A) Phosphatidylinositol turnover is expressed as dpm ~H-inositol p h o s p h a t e s / m g tissue. Bars represent the mean + S.E.M. of 4-6 animals. Galanin incubation produced a significant increase in phosphatidylinositol turnover (*, P < 0.05 by analysis of variance) at both concentrations tested. (B) Phosphatidylinositol turnover in the atrium is expressed as the percent of the maximal response produced by galanin over the concentration range within a given experiment. Bars represent the mean + S.E.M. of 4-6 experiments.
89
results expressed as dpm 3H-inositol p h o s p h a t e s / mg frozen tissue. The average weight for the muscle strips was between 10-15 mg. For cyclic nucleotide measurements, single strips of either atrium or ventricle were first equilibrated for 30 min following dissection in control solution. The strips were then rinsed with fresh solution, with a final volume of 1.5 ml. Galanin (10 -7 M) or isoproterenol (2 ~ M ) was added to the solution and the tissue was incubated for either 2 or 5 min at room temperature. The reaction was stopped by rapidly freezing the tissue in liquid nitrogen. The tissue was then weighed and homogenized in acidified ethanol (0.2 N HCI, 65% EtOH). The homogenate was centrifuged at 2000 × g for 5 min at 4°C and the supernate was lyophilized and stored at - 7 0 ° C for later analysis. Cyclic AMP levels were determined in duplicate for each sample using a scintillation proximity assay (Amersham, RPA.538) and cyclic G M P levels were determined in duplicate by radioimmunoassay (Amersham, RPA.525). Results are expressed as the percent of the basal level of cyclic nucleotide.
Results and Discussion
Incubation of either isolated atrial or ventricular strips with galanin produced a significant increase in phosphatidylinositol turnover (Fig. la) and this stimulation was concentration dependent (Fig. lb). Accumulation of 3H-inositol phosphates increased with concentrations of galanin up to 10 -8 M, but at higher concentrations there was a decrease in the response which may reflect a concentration-dependent desensitization of the galanin receptor. Inhibition of twitch-tension development in isolated atrial and ventricular strips was previously observed with galanin concentrations of 10 -7 M to 10 -6 M [11]. In addition, voltage recordings from parasympathetic neurons of the cardiac ganglion showed a desensitization of the galanin-induced hyperpolarization with repeated application in 14% of the cells [5]. Similar results were also seen in recordings from atrial myocytes (Konopka and Parsons, unpublished observations). The ECs0 for the galanin stimulation
TABLE I
The effect of galanin and isoproterenol on cyclic nucleotide levels in the mudpuppy heart Atrium
Ventricle
Cyclic AMP Cyclic GMP
Cyclic AMP
Galanin 95%_+10 92%_+20 (5) (6) Iso 290%_+66* 96%+13
(5) Galanin 202%_+24*
+ Iso (4)
(6)
Cyclic GMP
96%_+10 86%_+17 (6) (5) 548%_+134* 91%+16
(6)
(5)
116%_+17 576%_+116*
74%_+25
(6)
(5)
(6)
The values given are the percent of basal levels of cyclic AMP or cyclic GMP (mean_+ S.E.M.) of n animals, given in parentheses. Significant differences as determined by analysis of variance are indicated by the asterisk (P < 0.05). The basal cyclic AMP levels were as follows: atrium, 156.6_+23.8 fmol/mg tissue (n = 11) and ventricle, 172.6_+ 39.8 fmol/mg tissue (n = 10). The basal cyclic GMP levels were: atrium 449_+7.2 fmol/mg tissue ( n = 6 ) and ventricle, 23.5+4.9 fmol/mg tissue (n = 4).
of phosphoinositide turnover was estimated to be approximately 7.5 nM, a value consistent with those reported for galanin-induced changes in other systems [1,9]. Thus, the concentration of galanin necessary to produce cardio-inhibition correlates with that which stimulates phosphatidylinositol turnover. The effect of galanin on the levels of cAMP and cGMP in both the atria and the ventricles was determined in the resting state and with adrenergic stimulation. Previous studies had demonstrated a muscarinic inhibition of cyclic AMP in the heart with adrenergic stimulation [4]. Galanin had no effect on basal levels of either cyclic AMP or cyclic GMP (Table I) in either atrium or ventricle. As shown previously in cardiac tissue [11], isoproterenol transiently increased the levels of cyclic AMP in both the atrium and the ventricle at early time points (2 min), but this increase disappeared by 5 min of incubation (data not shown). Isoproterenol had no effect on basal cyclic GMP levels. Co-incubation with galanin did not produce any significant change in the isoproterenol-induced increase in cyclic AMP, nor did it affect cyclic GMP levels in adrenergically-stimulated preparations.
90
Our results demonstrate that galanin produced a concentration-dependent stimulation of phosphatidylinositol turnover in the mudpuppy myocardium but did not alter either basal or catecholamine-stimulated cyclic nucleotide levels in atrial or ventricular preparations. Galanin exerts an inhibitory effect on many different cell types. However, this inhibition has previously been associated with either an inhibition of phosphoinositide turnover such as in hippocampal cells [9] or a decrease in adenylate cyclase activity as shown for insulin-secreting cells [1] and cortical cells [8]. Thus, the alteration of intracellular second messenger levels by galanin in mudpuppy cardiac tissue is different from that reported for other systems. Acknowledgements We would like to thank Drs. Edith Hendley, Karen Braas and Victor May for critical review of the manuscript. This work was supported by NIH Grants NS-23978 to R.L.P. and NS-08580 to J.C.H. References 1 Amiranoff, B., Lorinet, A.-M., Lagny-Pourmir, I. and Laburthe, M., Mechanism of galanin-inhibited insulin release, Eur. J. Biochem., 177 (1988) 147-152.
2 Axelsson, M. and Nilsson, S.. ('ontrol of the heart in thc mudpuppy, Necturus maculosus, Exp. Biol.. 44 (1985) 229-239. 3 Berridge, M.J., Dawson, R.M.C., Downes, C.P., Hcslop, J.P. and lrvine, R.F., Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides, Biochem. J., 212 (1983)473-482. 4 Hartzcll, H.C., Regulatkm of cardiac ion channels by catecholamines, acetylcholinc, and second messenger systems, Prog. Biophys, Mol. Biol., 52 (1988) It~5-248. 5 Konopka, L,M., McKeon, T.W. and Parsons, R.L., Galanin-induced hyperpolarization and decreased membrane excitability of neurones in mudpuppy cardiac ganglia, J. Physiol., 410 (1989)1{17. 6 McKeon, T.W. and Parsons, R.L., Galanin immunoreactivity in the mudpuppy cardiac ganglion, J. Auton. Nerv. Syst., 31 (1990) 135-140. 7 Merriam, L.A., Konopka, L.M. and Parsons, R.L., Bethanechol and galanin initiate hyperpolarizatkm of mudpuppy atrial muscle cells by different mechanisms, Soc. Neurosci. Abstr., 15 (1989) 219. 8 Nishibori, M.. Oishi, R., ltoh, Y. and Saeki, K., Galanin inhibits noradrenaline-induced accumulation of cyclic AMP in the rat cerebral cortex, J. Neurochem.. 51 (1088) 1953-1955. 9 Palazzi, E., Fisone, G., Hokfelt, T., Bartfai, T. and Consolo, S., Galanin inhibits the muscarinic stimulation of phosphoinositide turnover in rat ventral hippocampus, Eur. J. Pharmacol.. 148 (1988) 479-480. 10 Parsons, R.L., Neel, D.S., Konopka, L.M. and McKeon, T.W., The presence and possible role of a galanin-like peptide in the mudpuppy heart, Neurosci, 29 (1989) 740759. 11 Tsien, R.W. Cyclic AMP and contractile activity in heart, Adv. Cyclic Nucleotide Res., 8 (1977) 363-420.
87
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01302
Galanin stimulates phosphatidylinositol turnover in cardiac tissue of the mudpuppy Jean C. Hardwick and R o d n e y L. Parsons Department of Anatomy and Neurobiology, College of Medicine, Unit'ersityof Vermont, Burlington, Vermont, USA (Received 17 March 1992) (Revision received 12 May 1992) (Accepted 19 May 1992)
Key words: Galanin; Inositol phosphate; Cardiac muscle; Cyclic AMP; Cyclic GMP Abstract A galanin-like peptide has been found in the parasympathetic fibers innervating the mudpuppy myocardium and direct application of galanin produces hyperpolarization of atrial myocytes and a decrease in twitch tension. In the present study, atrial and ventricular strips were incubated with galanin and then evaluated for changes in either phosphatidylinositol turnover or cyclic nucleotide levels. Galanin caused a significant and concentration-dependent increase in phosphatidylinositol turnover in both atrial and ventricular tissue. However, galanin had no effect on cyclic AMP or cyclic GMP levels in either basal or adrenergically-stimulated preparations. These results suggest that the galanin-induced cardio-inhibition in the mudpuppy may be mediated, at least in part, by a change in phosphatidylinositol turnover.
Introduction
In 1985, Axelsson and Nilsson demonstrated that vagal stimulation of the mudpuppy (Necturus maculosus), in the presence of cholinergic antagonists, produced cardio-inhibition [2]. These authors suggested that another unidentified neurotransmitter, perhaps a neuropeptide, was colocalized with acetylcholine and released from parasympathetic nerve terminals. Subsequently, we demonstrated, both biochemically and histochemically, that a galanin-like peptide is present in postganglionic parasympa-
Correspondence to: R.L. Parsons, Department of Anatomy and Neurobiology, Given Building, University of Vermont, Burlington, VT 05405, USA.
thetic fibers innervating the mudpuppy heart [6,10]. We also observed in isolated atrial and ventricular strips, a galanin-induced decrease in electrically stimulated twitch tension and a decrease in force and rate of spontaneous contractions in isolated septum/sinus venous preparations [10]. Local application of galanin directly onto muscle strands in septal or atrial whole mount preparations or to atrial myocytes hyperpolarized these cells [7,10]. From these results, it appears that a peptide similar to mammalian galanin is the previously unidentified neuropeptide which can produce non-cholinergic cardio-inhibition in the mudpuppy. The alteration in activity of target tissues by neuropeptides is commonly mediated through the activation of intracellular second messengers. Consequently, the present study was undertaken
to determine whether galanin activates second messengers in mudpuppy myocardium which might mediate the galanin-induced cardio-inhibition.
Materials and Methods Mudpuppies were killed by rapid decapitation, the heart removed and the atrial and ventricular regions dissected free. The atria were bisected and the ventricles cut into strips of similar size in a solution containing (in raM): 120 NaCI, 2.5 KCI, 3.6 CaCI 2, 1.0 Hepes, pH 7.3 at 20-22°C. Sections were then randomly distributed such that one or two strips of either atrial or ventricular tissue were loosely pinned in Sylgard-coated wells. To measure phosphatidylinositol turnover, isolated strips were incubated overnight at room temperature in normal Ringer solution containing 0.2 # M 3H-myo-inositol (New England Nuclear, 14-20 C i / m m o l ) to label the phospholipid
pool. Following this incubation, the [issue was washed with several ml of fresh solution and then incubated for 10 min in 1.5 ml of control solution containing 10 mM LiCI to inhibit inositol phosphatase. Galanin (porcine, Peninsula Laborat¢)ries or Sigma Chemical Co.) at concentrations ranging from I0 '~ to 10 +' M was added and the incubation continued for 20 rain at room temperature. The tissue was then immersed in liquid nitrogen, weighed and homogenized in acidified methanol (1: 100) in a ground glass homogenizer. The final volume of 2 ml was centrifuged at 2 0 0 0 x g for 11) min at 4°C and 1.4 ml of the resulting supernate was diluted with 5 ml of water for analysis of inositol phosphates. Total inositol phosphates were isolated by column chromatography, as described by Berridge et al. [3]. Samples were poured over l)owex X 1-8 formate resin and the inositol phosphates collected by elution with 0.1 M formic a c i d / l M ammonium formate solution. The eluate was then counted in a liquid scintillation counter and the
B
A CONIROL
50
tO
-7
100
M GALANIN
I 0 6M GALANIN
40
z o uS
30
c~
80
2D
o
6o
\\, I
I I
20
c3
10
I
"5
40
2~
~
20
x
ATRIA
VENTRICLE
9
!
i
i
-8
-7
-6
log {GALANIN]
Fig. 1. Galanin stimulation of phosphatidylinositol turnover in atrial and ventricular tissue. Atrial and ventricular strips were prelabeled with ~H-myo-inositol and then incubated for 20 rain with either control solution or galanin at the concentration indicated. (A) Phosphatidylinositol turnover is expressed as dpm ~H-inositol p h o s p h a t e s / m g tissue. Bars represent the mean + S.E.M. of 4-6 animals. Galanin incubation produced a significant increase in phosphatidylinositol turnover (*, P < 0.05 by analysis of variance) at both concentrations tested. (B) Phosphatidylinositol turnover in the atrium is expressed as the percent of the maximal response produced by galanin over the concentration range within a given experiment. Bars represent the mean + S.E.M. of 4-6 experiments.
89
results expressed as dpm 3H-inositol p h o s p h a t e s / mg frozen tissue. The average weight for the muscle strips was between 10-15 mg. For cyclic nucleotide measurements, single strips of either atrium or ventricle were first equilibrated for 30 min following dissection in control solution. The strips were then rinsed with fresh solution, with a final volume of 1.5 ml. Galanin (10 -7 M) or isoproterenol (2 ~ M ) was added to the solution and the tissue was incubated for either 2 or 5 min at room temperature. The reaction was stopped by rapidly freezing the tissue in liquid nitrogen. The tissue was then weighed and homogenized in acidified ethanol (0.2 N HCI, 65% EtOH). The homogenate was centrifuged at 2000 × g for 5 min at 4°C and the supernate was lyophilized and stored at - 7 0 ° C for later analysis. Cyclic AMP levels were determined in duplicate for each sample using a scintillation proximity assay (Amersham, RPA.538) and cyclic G M P levels were determined in duplicate by radioimmunoassay (Amersham, RPA.525). Results are expressed as the percent of the basal level of cyclic nucleotide.
Results and Discussion
Incubation of either isolated atrial or ventricular strips with galanin produced a significant increase in phosphatidylinositol turnover (Fig. la) and this stimulation was concentration dependent (Fig. lb). Accumulation of 3H-inositol phosphates increased with concentrations of galanin up to 10 -8 M, but at higher concentrations there was a decrease in the response which may reflect a concentration-dependent desensitization of the galanin receptor. Inhibition of twitch-tension development in isolated atrial and ventricular strips was previously observed with galanin concentrations of 10 -7 M to 10 -6 M [11]. In addition, voltage recordings from parasympathetic neurons of the cardiac ganglion showed a desensitization of the galanin-induced hyperpolarization with repeated application in 14% of the cells [5]. Similar results were also seen in recordings from atrial myocytes (Konopka and Parsons, unpublished observations). The ECs0 for the galanin stimulation
TABLE I
The effect of galanin and isoproterenol on cyclic nucleotide levels in the mudpuppy heart Atrium
Ventricle
Cyclic AMP Cyclic GMP
Cyclic AMP
Galanin 95%_+10 92%_+20 (5) (6) Iso 290%_+66* 96%+13
(5) Galanin 202%_+24*
+ Iso (4)
(6)
Cyclic GMP
96%_+10 86%_+17 (6) (5) 548%_+134* 91%+16
(6)
(5)
116%_+17 576%_+116*
74%_+25
(6)
(5)
(6)
The values given are the percent of basal levels of cyclic AMP or cyclic GMP (mean_+ S.E.M.) of n animals, given in parentheses. Significant differences as determined by analysis of variance are indicated by the asterisk (P < 0.05). The basal cyclic AMP levels were as follows: atrium, 156.6_+23.8 fmol/mg tissue (n = 11) and ventricle, 172.6_+ 39.8 fmol/mg tissue (n = 10). The basal cyclic GMP levels were: atrium 449_+7.2 fmol/mg tissue ( n = 6 ) and ventricle, 23.5+4.9 fmol/mg tissue (n = 4).
of phosphoinositide turnover was estimated to be approximately 7.5 nM, a value consistent with those reported for galanin-induced changes in other systems [1,9]. Thus, the concentration of galanin necessary to produce cardio-inhibition correlates with that which stimulates phosphatidylinositol turnover. The effect of galanin on the levels of cAMP and cGMP in both the atria and the ventricles was determined in the resting state and with adrenergic stimulation. Previous studies had demonstrated a muscarinic inhibition of cyclic AMP in the heart with adrenergic stimulation [4]. Galanin had no effect on basal levels of either cyclic AMP or cyclic GMP (Table I) in either atrium or ventricle. As shown previously in cardiac tissue [11], isoproterenol transiently increased the levels of cyclic AMP in both the atrium and the ventricle at early time points (2 min), but this increase disappeared by 5 min of incubation (data not shown). Isoproterenol had no effect on basal cyclic GMP levels. Co-incubation with galanin did not produce any significant change in the isoproterenol-induced increase in cyclic AMP, nor did it affect cyclic GMP levels in adrenergically-stimulated preparations.
90
Our results demonstrate that galanin produced a concentration-dependent stimulation of phosphatidylinositol turnover in the mudpuppy myocardium but did not alter either basal or catecholamine-stimulated cyclic nucleotide levels in atrial or ventricular preparations. Galanin exerts an inhibitory effect on many different cell types. However, this inhibition has previously been associated with either an inhibition of phosphoinositide turnover such as in hippocampal cells [9] or a decrease in adenylate cyclase activity as shown for insulin-secreting cells [1] and cortical cells [8]. Thus, the alteration of intracellular second messenger levels by galanin in mudpuppy cardiac tissue is different from that reported for other systems. Acknowledgements We would like to thank Drs. Edith Hendley, Karen Braas and Victor May for critical review of the manuscript. This work was supported by NIH Grants NS-23978 to R.L.P. and NS-08580 to J.C.H. References 1 Amiranoff, B., Lorinet, A.-M., Lagny-Pourmir, I. and Laburthe, M., Mechanism of galanin-inhibited insulin release, Eur. J. Biochem., 177 (1988) 147-152.
2 Axelsson, M. and Nilsson, S.. ('ontrol of the heart in thc mudpuppy, Necturus maculosus, Exp. Biol.. 44 (1985) 229-239. 3 Berridge, M.J., Dawson, R.M.C., Downes, C.P., Hcslop, J.P. and lrvine, R.F., Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides, Biochem. J., 212 (1983)473-482. 4 Hartzcll, H.C., Regulatkm of cardiac ion channels by catecholamines, acetylcholinc, and second messenger systems, Prog. Biophys, Mol. Biol., 52 (1988) It~5-248. 5 Konopka, L,M., McKeon, T.W. and Parsons, R.L., Galanin-induced hyperpolarization and decreased membrane excitability of neurones in mudpuppy cardiac ganglia, J. Physiol., 410 (1989)1{17. 6 McKeon, T.W. and Parsons, R.L., Galanin immunoreactivity in the mudpuppy cardiac ganglion, J. Auton. Nerv. Syst., 31 (1990) 135-140. 7 Merriam, L.A., Konopka, L.M. and Parsons, R.L., Bethanechol and galanin initiate hyperpolarizatkm of mudpuppy atrial muscle cells by different mechanisms, Soc. Neurosci. Abstr., 15 (1989) 219. 8 Nishibori, M.. Oishi, R., ltoh, Y. and Saeki, K., Galanin inhibits noradrenaline-induced accumulation of cyclic AMP in the rat cerebral cortex, J. Neurochem.. 51 (1088) 1953-1955. 9 Palazzi, E., Fisone, G., Hokfelt, T., Bartfai, T. and Consolo, S., Galanin inhibits the muscarinic stimulation of phosphoinositide turnover in rat ventral hippocampus, Eur. J. Pharmacol.. 148 (1988) 479-480. 10 Parsons, R.L., Neel, D.S., Konopka, L.M. and McKeon, T.W., The presence and possible role of a galanin-like peptide in the mudpuppy heart, Neurosci, 29 (1989) 740759. 11 Tsien, R.W. Cyclic AMP and contractile activity in heart, Adv. Cyclic Nucleotide Res., 8 (1977) 363-420.